Alcohol dehydrogenase mutant and use thereof

A technology of alcohol dehydrogenase and mutants, which is applied in the field of enzyme engineering and can solve the problems of large mutant library and dependence on high-throughput screening methods

Active Publication Date: 2019-02-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practice, due to the large size of the mutant library constructed, it relies on high-throughput screening methods

Method used

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  • Alcohol dehydrogenase mutant and use thereof
  • Alcohol dehydrogenase mutant and use thereof
  • Alcohol dehydrogenase mutant and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0073] 1. Semi-rational design of alcohol dehydrogenase LkADH

[0074] 1.1 Site-directed mutations at positions 147 and 202

[0075] (1) F147L, F147I, F147M, F147C, A202L, A202I, A202V mutants

[0076] Using the recombinant plasmid Pet-30a(+)-LkADH connected with the alcohol dehydrogenase LkADH gene as a template, a pair of forward and reverse primers F-Primer / R-Primer (Table 1, 2) containing mutation sites were designed. The 147th and 202nd positions were subjected to site-directed mutagenesis by PCR.

[0077] Table 1 147-site site-directed mutagenesis primer sequence

[0078]

[0079]

[0080] Table 2 202 site-directed mutagenesis primer sequences

[0081]

[0082] (2) Obtaining F147L / A202L, F147I / A202L mutants:

[0083] A pair of forward and reverse primers F-Primer / R-Primer (Table 3), performing site-directed mutagenesis by PCR.

[0084] Table 3 147, 202 combination mutation primer sequence

[0085]

[0086] As shown in Table 4, site-directed mutations w...

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Abstract

The invention discloses an alcohol dehydrogenase mutant and a use thereof. The alcohol dehydrogenase mutant is shown in the formula (1) and / or (2). (1) Phenylalanine at 147th of an amino acid sequenceshown in the formula of SEQ ID NO.1 is replaced by leucine, isoleucine, methionine or cysteine. (2) Alanine at 202th of the amino acid sequence shown in the formula of SEQ ID NO.1 is replaced with leucine, isoleucine or valine. A semi-rational design is used to modify the alcohol dehydrogenase LkADH derived from Lactobacillus kefir so that a plurality of alcohol dehydrogenase mutants are obtained. The alcohol dehydrogenase mutant has the characteristics of high enzyme activity, can efficiently catalyze t-butyl 6-chloro-3, 5-dicarbonylcaproate to prepare t-butyl (S)-6-chloro-5-hydroxy-3-carbonylcaproate and has a great significance for the production of statin drug intermediates.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an alcohol dehydrogenase mutant and its application. Background technique [0002] (S)-6-Chloro-5-hydroxy-3-oxoylhexanoic acid tert-butyl ester ((S)-CHOH) is a very important chiral intermediate, mainly used in the synthesis of HMG-CoA reductase inhibitors, which further The product of asymmetric reduction can not only synthesize atorvastatin calcium (Lipitor) through steps such as dehalogenation, cyano group, hydroxyl protection, but also rosuvastatin calcium (can be synthesized by hydroxyl protection, acylation and hydrolysis reactions). Both are the best-selling blood lipid-lowering drugs in the world. [0003] At present, (S)-CHOH is mainly synthesized through two synthetic routes, one is to use ethyl 4-chloro-3-carbonylbutyrate as a substrate, and asymmetrically synthesize (S)-4-chlorobutyrate by chemical or biological methods. -Ethyl 3-hydroxybutyrate, and reac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62
CPCC12N9/0006C12N15/70C12P7/62C12Y101/01001
Inventor 吴坚平陈方徐刚杨立荣
Owner ZHEJIANG UNIV
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